The purpose of this proposal is to analyze the molecular mechanisms that govern integrin-ligand binding. Current analyses based on the solution of a crystal structure for alphavbeta3 have focused the conformational changes related to ligand binding. These conformational changes may be important for signaling as well as control of ligand binding. Nevertheless, in the context of the whole cell additional factors appear to contribute to the regulation of integrin-ligand binding and the strength of adhesion. The existence other factors have been Tong recognized as "avidity ". Although elements of the process, such as integrin clustering, have been associated with avidity, there is no clear mechanism to explain how these factors modulate adhesion. Part of the problem lies in the measurement of adhesion. The development of the spinning disc and chemical cross-linking methods provided tools to analyze the integrin-ligand binding interaction in the cell-substrate interface or contact zone. In this proposal, we plan to further develop the analysis to discriminate between measuring the number of integrin-ligand bonds that are formed and the strength of those bonds. These tools will be combined with laser tweezers and total internal reflection fluorescence microscopy to investigate a5b1-flbronectin binding in the contact zone. Results obtained in the previous funding period have shown that the binding parameters in the contact zone are very different from the binding of soluble ligand and generate a case in which the proportion of bound integrin can be regulated by the local density. Following the initial binding of alpha5beta1 to fibronectin, a5b1 becomes linked to the actin cytoskeleton which is in turn tensioned by the action of myosin motors. This will apply tension to the alpha5beta1-fibronectin bond (only as a post ligand-binding event). Results from the previous funding period show that there is a change in the alpha5beta1-fibronectin binding interface that occurs when tension is applied and can be detected by specific chemical cross-linking of alpha5beta1 to fibronectin.